Hemoglobin (Hb) is the major constituent of the erythrocyte which carries oxygen from the lungs throughout the body. When contained in red blood cells, Hb exists as a tetramer structure composed of two oxygen linked .alpha..beta. dimers each having a molecular weight of about 32 Kd. Each .alpha. and .beta. subunit of each dimer has a protein chain and a heme molecule. The sequences of the .alpha. and .beta. protein chains are known. Hb is a potentially useful blood substitute for transfusions, and has been proposed as a reagent to trap nitric oxide in septic shocks, and to modulate tissue oxygenation during the radiation therapy of cancer. Recombinant DNA technology also has afforded the generation of modified Hb with oxygen affinities modulated for special needs of individual therapeutic applications.
The potential use of Hb as blood substitutes in transfusions or other therapeutic applications, however, has been hampered by the short circulation half-life of Hb. In solution outside of the red blood cell, Hb readily dissociates from its tetrameric form into its dimers and even monomers, which are rapidly filtered through the kidneys. Accordingly, a multitude of methods for crossbridging Hb (e.g. bifunctional modification) and other means for increasing the hydrodynamic volume of Hb (e.g. monofunctional decoration) have been devised to limit or prevent the extravasation of Hb.
Simon, S. R. and Konigsberg, W. H. reported the use of bis(N-maleimidomethyl) ether (BME) to generate intra-crosslinked Hb (Proc. Natl. Acad. Sci. 56:749-756 (1966). Bunn, H. F., et al. later reported that BME crosslinked Hb increased the half-life of Hb four-fold when infused into rats and dogs (J. Exp. Med. 129:909-924 (1969)). However, the crosslinking of Hb with (BME) resulted in a concomitant increase in the oxygen affinity of Hb which prevented its use as a potential Hb-based oxygen carrier.
Xue, H. and Wong, J. T.-F. described many of the current methods for crosslinking Hb (Methods in Enzymology 231:308-322 (1994)). These include the use of dextran, hydroxyethyl starch, inulin, polyvinylpyrrolidone, and polyethylene glycol as crosslinkers for Hb. Other crosslinkers include glutaraldehyde (MacDonald, S. L. and Pepper, D. S. Methods in Enzymology 231:287-308 (1994)); bis(3,5-dibromosalicyl) fumarate (Walder, R. Y., et al. Methods in Enzymology 231:274-281 (1994)); acyl phosphate esters (Kluger, R., et al. Art. Cells, Blood Subs., and Immob. Biotech. 22(3):415-428 (1994) and U.S. Pat. No. 5,334,707); bissulfosuccinimidyl esters of aliphatic dicarboxylic acids (Manjula, B. N., et al. Art. Cells, Blood Subs., and Immob. Biotech. 22(3):747-752 (1994)); and benzenepentacarboxylate (U.S. Pat. No. 5,349,054).
Nho, K, et al. described the monofunctional decoration of hemoglobin with polyethylene glycol (Art. Cells, Blood Subs., and Immob. Biotech. 22(3):795-803 (1994)). Similar modification of the hemoglobin molecule are described in U.S. Pat. Nos. 4,670,417, 5,234,903, and 5,312,808.
Most of the known compounds used to modify Hb are difficult to synthesize, do not modify Hb in an efficient manner, cannot be manipulated quantitatively to form the desired modification, or lower or raise the oxygen affinity of the modified hemoglobin. Accordingly, there exists a need for new compounds which are easily synthesized, modify hemoglobin in an efficient and focused manner, and do not substantially affect the oxygen affinity of the modified hemoglobin. The present invention satisfies this need.